急性肺损伤小鼠肺组织CCL20的表达及其在巨噬细胞炎症反应中的作用

Expression of CCL20 in mouse lung tissue with acute lung injury and its potential role in macrophage inflammatory response

目的观察趋化性细胞因子CCL20在急性肺损伤小鼠肺组织中的表达,研究气道及肺泡上皮细胞CCL20表达的调控及其在巨噬细胞炎症反应中的作用。方法采用气道滴注脂多糖(LPS)建立小鼠急性肺损伤模型,通过HE染色观察小鼠肺组织炎症反应,免疫组化染色观察小鼠肺组织CCL20的表达,同时利用qRT-PCR检测肺组织中CCL20 mRNA表达水平。并设置细胞实验,分别以LPS、TNF-α和IL-1β刺激人气道上皮细胞16HBE和肺泡上皮细胞A549,qRT-PCR检测CCL20 mRNA表达水平;并以CCL20刺激小鼠单核巨噬细胞RAW264.7,qRT-PCR检测炎症因子单核细胞趋化蛋白-1(MCP-1)、IL-6、环氧合酶2 mRNA表达水平。结果与对照组比较,造模组小鼠肺组织炎症反应显著加重;免疫组化显示肺组织CCL20蛋白的表达在气道上皮细胞更为明显;造模组小鼠肺组织CCL20 mRNA表达水平较对照组升高(P<0.01)。同时发现,LPS、TNF-α、IL-1β刺激后人气道上皮细胞16HBE及肺泡上皮细胞A549 CCL20 mRNA表达水平较对照组均升高(均P<0.01),重组CCL20蛋白刺激4 h后小鼠单核巨噬细胞RAW264.7 MCP-1 mRNA表达水平较对照组升高(P<0.05)。结论气道上皮来源的CCL20在急性肺损伤中表达水平显著升高,其表达受LPS、TNF-α和IL-1β多种炎症因子的调控,CCL20在巨噬细胞介导的炎症反应中发挥了一定作用,可为未来急性肺损伤的诊治靶点研究提供新的思路。

Objective To observe the expression of chemokine CCL20 in acute lung injury in mice and to clarify the CCL20 expression regulation in airway and alveolar epithelial cells,and its potential role of CCL20 in macrophage inflammatory response in vitro.Methods Airway lipopolysaccharide(LPS)infusion was used to establish an acute lung injury mice model,HE staining was used to observe the inflammatory response in lung tissues,immunohistochemistry was adopted to observe the localization and expression of CCL20 in lung tissue,qRT-PCR was adopted to observe CCL20 mRNA level in lung tissue homogenate.In vitro study,LPS,TNF-αor IL-1βwere used to stimulate human airway epithelial cells 16HBE and alveolar epithelial cell A549,qRT-PCR was adopted to observe CCL20 mRNA level in these cells,CCL20 was used to stimulate mouse mononuclear macrophage RAW264.7,and the mRNA expression levels of monocyte chemotactic protein-1(MCP-1),IL-6,and cyclooxygenase-2(COX-2)were detected by qRT-PCR.Results Compared with the control group,the inflammatory response of lung tissue was more severe in LPS-induced acute lung injury mice model.Immunohistochemistry showed the expression of CCL20 protein in lung tissues was basically located in airway epithelial cells.The level of CCL20 mRNA in the lung tissue homogenate of the model group was significantly higher than control group(P<0.01).The expression of CCL20 mRNA in alveolar epithelial cells 16HBE and alveolar epithelial cell A549 stimulated by LPS,TNF-αor IL-1βwere significantly higher than control group(all P<0.01).After 4 h stimulation of recombinant protein CCL20,the level of MCP-1 mRNA expression in mouse mononuclear macrophages RAW264.7 significantly increased(P<0.05).Conclusion Airway epithelium-derived CCL20 is up-regulated in lung injury,and its expression is regulated by various inflammatory mediators including LPS,TNF-αand IL-1β.CCL20 plays a certain role in macrophage-mediated inflammatory response,providing a new idea for the future diagnosis and treatment of acute lung injury.